The present invention is directed to a system for co-culturing bacteria and eukaryotic cells. The apparatus allows bacteria that are in a controlled state of growth to be perfused over immobilized eukaryotic cells. In addition, the invention includes a variety of methods for studying the attachment and invasion of host cells by bacteria.
At present, most protocols for studying the interaction between bacteria and host tissue involve growing the bacteria in batch culture, placing them onto a monolayer of eukaryotic cells, and then treating the monolayer to study either attachment or invasion. However, growth rate cannot be controlled using batch culture methods. Since expression of bacterial cell surface proteins is essential for their invasion of host cells, and since it is known that such expression can be altered by growth rate, this is a serious drawback (see Paoletti, et al., Infect. Immun. 64:1220-1226 (1996); Ross, et al., J Bacteriol. 181:5389-5394 (1999)).
Although methods for growing bacteria under defined steady state conditions are known and systems for perfusing cultured eukaryotic cells with media have been disclosed (see e.g., U.S. Pat. No. 5,565,353; U.S. Pat. No. 5,693,537; Hulten et al., Agents Chemother. 40:2727-2731 (1996)), these have not been combined into an effective system for studying the interactions between invasive bacteria and their hosts. The development of such a system would have a number of important applications. First, the ability to separate invasive bacteria from their non-invasive counterparts should aid researchers in determining the mechanisms involved in the invasion process and may lead to the development of new therapeutic strategies for treating bacterial diseases. The technology could also be used to screen for therapeutic agents that act by preventing bacterial attachment and invasion. In addition, defining the growth conditions which increase bacterial invasiveness would allow cells to be grown that are especially well suited to the development of antibodies for diagnostic or therapeutic purposes.
The present invention is based upon the discovery that growth conditions affect the ability of bacteria to invade host cells. This has led the inventors to develop a system for combining previously unrelated methods for growing bacterial and eukaryotic cells. The key element of the system is that bacteria are grown at a defined rate under steady state conditions and used to perfuse eukaryotic cells that are attached to a solid support, thus preserving a defined bacterial state at all times during their interaction with eukaryotic cells. The apparatus and methods developed should be useful to researchers studying bacterial diseases and should aid in the development of new therapeutic and diagnostic procedures.
In its first aspect, the invention is directed to an apparatus in which eukaryotic and bacterial cells are co-cultured under different conditions. The apparatus has a fermentor in which bacteria are grown at a fixed rate under steady state conditions. The fermentor must have at least one inlet port for receiving materials needed to maintain bacterial growth (e.g., growth medium). By altering the availability of one or more nutrients, e.g., glucose, the rate at which the bacteria grow can be controlled. Thus, the inlet port will typically be connected via tubing to a reservoir of nutrient medium and means for actively delivering this medium to the port will be present. The fermentor may also include a second inlet port for receiving materials and which may be connected to a second reservoir. For example, the second inlet port might be used to deliver a solution to help control the pH at which cells are grown. In addition, the fermentor should have at least one outlet port through which fluid from inside the fermentor, comprised of growth medium and bacteria, can pass. It should also have means for mixing the fluid it contains, e.g., a rotating paddle mixer.
The apparatus includes a culture vessel containing eukaryotic cells attached to a solid support. This vessel may take the form of a tissue culture flask, dish, or multiwell plate in which cells are attached either directly to the walls of the vessel or to a matrix used to coat the walls. It may also take the form of a column that has been packed with a porous support (e.g., beads or membranes) on which cells have been grown or even roller bottles adapted to be used with the system. The culture vessel must have at least one inlet port connected, usually by polymeric tubing, to the outlet port of the fermentor. Means for moving fluid from the fermentor""s outlet port to the inlet port of the culture vessel must also be available to allow the eukaryotic cells to be perfused with bacteria from the fermentor. Any type of pump compatible with biological systems, e.g., a peristaltic pump, can be used for this purpose. In addition, the culture vessel must have at least one outlet port for removing fluid. Since the eukaryotic cells are immobilized by being attached to a solid support, they remain within the culture vessel as fluid is removed. Although such removal may take place through the outlet port passively in response to fluid being pumped in, it is preferred that means for actively extracting fluid, e.g., a pump, be present.
In a preferred embodiment, the fermentor of the apparatus described above contains a second outlet port for removing fluid which, unlike the first outlet port, is not connected to the culture vessel. The second outlet is used to maintain a constant volume and allows the rate at which nutrient material enters into the fermentor to be altered without the need for changing the rate at which eukaryotic cells are perfused.
In another aspect, the invention is directed to a method for assaying bacteria for their ability to attach to and invade eukaryotic cells. This is accomplished by growing the bacteria in a continuous culture fermentor under steady state conditions and simultaneously perfusing the growing bacteria over eukaryotic cells that are attached to a solid support. The invasiveness of the bacteria can then be determined by lysing the eukaryotic cells and counting the number of colony forming units of bacteria. This can be done either directly or, the culture flask can be removed from the apparatus, further incubated if desired, and then inspected microscopically. Other means for determining the extent to which bacteria have attached to and invaded eukaryotic cells are also compatible with the invention. The apparatus described above is particularly well suited for this assay although alternative systems can be employed if desired.
The ability to separate bacteria that attach to eukaryotic cells from those that do not is of interest to researchers studying bacterial diseases. The apparatus described above lends itself to a method for accomplishing such a separation. Again, bacteria are grown in a continuous culture fermentor under steady state conditions and allowed to perfuse eukaryotic cells attached to a solid support. When the perfusate is removed from the cells, unattached bacteria are eliminated and those attached to the eukaryotic cells remain behind. After washing, e.g., with bacteria-free medium, the attached bacteria can either be studied directly or grown in suspension. In some instances, it may also be desirable to collect the perfusate from the culture vessel in order to obtain bacteria that do not attach to eukaryotic cells. This could be useful for scientists wanting to compare the characteristics of invasive bacteria with their non-invasive counterparts.
The invention also includes a method of assaying a test compound for its ability to block the invasion of eukaryotic cells by bacteria, or to clear invaded bacteria from infected cells. Similar to the methods described above, bacteria are grown in a continuous culture fermentor under steady state conditions and then used to perfuse eukaryotic cells that are attached to a solid support. The number of bacteria that invade the eukaryotic cells is determined and then this entire process is repeated in the presence of the test compound. A reduction in the number of eukaryotic cells invaded would be an indication that the test compound acts as a blocking agent or is effective as an intracellular antibacterial agent, and has potential use as a therapeutic.
In another aspect, the basic procedures described above can be adapted to a method of selecting a growth rate at which the invasiveness of bacteria for eukaryotic cells is increased. Cells of increased invasiveness are of interest to scientists studying bacterial diseases as well as to those trying to develop antibodies that can be used for either therapeutic or diagnostic purposes. The method is performed by growing bacteria under steady state conditions at a first growth rate, perfusing these bacteria over eukaryotic cells as described above, and then determining the extent to which the cells are invaded. This process is then repeated at a second growth rate to determine which conditions lead to the most invasion. By performing the process several times, optimized conditions for growing bacteria can be determined.